鸽小肠上皮细胞的分离培养与鉴定

doi:10.3969/j.issn.1004-1524.2021.02.07

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (2): 239-247.DOI: 10.3969/j.issn.1004-1524.2021.02.07

鸽小肠上皮细胞的分离培养与鉴定

刘磊¹^,²(), 王讯¹^,²^,^*(), 罗毅¹^,², 梁继元¹^,², 颜培祺¹^,², 李昕怡¹, 刘薇¹, 刘辰恺¹, 李佳佳¹

1.四川农业大学动物科技学院,四川成都 611130
2.畜禽遗传资源发掘与创新利用四川省重点实验室,四川成都 611130

收稿日期:2020-06-20 出版日期:2021-02-25 发布日期:2021-02-25
通讯作者: 王讯
作者简介:*王讯,E-mail: xunwang@sicau.edu.cn
刘磊(1995—),男,山西繁峙人,硕士研究生,研究方向为鸽遗传育种。E-mail: 470578250@qq.com
基金资助:
四川农业大学双支计划(1921993255)

Isolation, culture and identification of pigeon intestine epithelium cells

LIU Lei1¹^,²(), WANG Xun¹^,²^,^*(), LUO Yi¹^,², LIANG Jiyuan¹^,², YAN Peiqi¹^,², LI Xinyi¹, LIU Wei¹, LIU Chenkai¹, LI Jiajia¹

1. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
2. Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Chengdu 611130, China

Received:2020-06-20 Online:2021-02-25 Published:2021-02-25
Contact: WANG Xun

摘要/Abstract

摘要：

本试验旨在建立鸽小肠上皮细胞体外分离培养和鉴定的方法体系,为研究鸽小肠上皮细胞的营养转运吸收、细胞增殖代谢等机制提供原代细胞模型。采用胶原酶消化法和组织块贴壁法分离鸽小肠上皮细胞,并通过相差消化法对其纯化,最后运用免疫荧光分析和基因表达分析鉴定鸽小肠上皮细胞。结果表明,2种方法均可成功分离培养鸽小肠上皮细胞,细胞呈铺路石状排列;细胞角蛋白8免疫荧光染色呈阳性;紧密连接蛋白基因CLDN-3和CLDN-4均有表达;细胞生长曲线呈“S”形。综上所述,本试验成功建立了鸽小肠上皮细胞体外分离培养体系,为后续开展雏鸽肠道营养研究提供了细胞模型。

关键词: 鸽, 小肠上皮细胞, 原代培养, 细胞鉴定

Abstract:

To establish a set of method to isolate, culture and identify pigeon intestine epithelium cells, and provide a primary cell model to research the mechanism of nutrient transportation and absorption, cell proliferation and metabolism in vitro, pigeon intestine epithelium cells were isolated by collagenase digestion and tissue explants adherent method. Isolated pigeon intestine epithelium cells were identified by immunofluorescence analysis and PCR analysis. The results showed that pigeon intestine epithelium cells were successfully isolated by using collagenase digestion and tissue explants adherent method. The isolated cells were arranged in the shape of paving stones. Cytokeratin 8 immunofluorescence staining was positive, and both CLDN-3 and CLDN-4 gene of tight junction proteins were expressed. The cell growth curve was S shape. In summary, we successfully established a set of method to isolate, culture and identify pigeon intestine epithelium cells, and provided a cell model for the further research on intestinal nutrition of pigeon squabs.

Key words: pigeon, intestine epithelium cells, primary culture, cell identification

中图分类号:

S836

刘磊, 王讯, 罗毅, 梁继元, 颜培祺, 李昕怡, 刘薇, 刘辰恺, 李佳佳. 鸽小肠上皮细胞的分离培养与鉴定[J]. 浙江农业学报, 2021, 33(2): 239-247.

LIU Lei1, WANG Xun, LUO Yi, LIANG Jiyuan, YAN Peiqi, LI Xinyi, LIU Wei, LIU Chenkai, LI Jiajia. Isolation, culture and identification of pigeon intestine epithelium cells[J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 239-247.

图/表 7

表1 RT-PCR引物序列

Table 1 Sequence of RT-PCR primers

基因 Gene	引物序列(5'→3') Primer sequence (5'→3')	NCBI登录号 NCBI accession No.	产物长度 Product length/bp
CLDN-3	F:GTGCAAGGTCTACGACTCCC R:TGAGGTTCATGATGGCGGAG	XM_005515008.2	212
CLND-4	F:CTTCTCTGAGACACCACCCG R:CAGGTTTGCTGTCCCCATCA	XM_021282730.1	184
β-actin	F:GTGGATCAGCAAGCAGGAGT R: TCATCACAAGGGTGTGGGTG	XM_005504502.2	101

图1 胶原酶消化法获得的鸽IECS形态观察 A,培养至1 d的细胞;B,培养至3 d的细胞;C,培养至5 d的细胞;D,培养至7 d的细胞。100×。

Fig.1 Morphological observation of pigeon intestine epithelium cells obtained by collagenase digestion A, Cells cultured for 1 day; B, Cells cultured for 3 days; C, Cells cultured for 5 days; D, Cells cultured for 7 days. 100×.

图2 组织块贴壁法获得的鸽IECS形态观察 A,组织块贴壁1 d后的细胞;B,组织块贴壁3 d后的细胞;C,组织块贴壁5 d后的细胞;D,组织块贴壁7 d后的细胞。A、B, 40×;C、D, 100×。

Fig.2 Morphological observation of pigeon intestine epithelium cells obtained by tissue block adherence method A, Explant culture of cells for 1 day; B, Explant culture of cells for 3 days; C, Explant culture of cells for 5 days; D, Explant culture of cells for 7 days. A、B, 40×; C、D, 100×.

图3 胰酶纯化前后鸽IECS形态观察 A,未纯化的细胞;B,纯化后的细胞。100×。

Fig.3 Morphological observation of pigeon intestine epithelium cells before and after purification by pancreatin A, Unpurified cells; B, Purified cells. 100×.

图4 鸽IECS生长曲线

Fig.4 Growth curve of pigeon intestine epithelium cells

图5 鸽IECS角蛋白8免疫荧光染色 A,明场下细胞形态;B,细胞角蛋白8免疫荧光染色;C,细胞核DAPI染色;D,B图与C图的融合。400×。

Fig.5 Cytokeratin-8 immunofluorescence staining of pigeon intestine epithelium cells A, Cell morphology under bright field; B, Immunofluorescence staining of cytokeratin-8; C, Nucleus stained by DAPI; D, Merged figure of B and C. 400×.

图6 IECS基因表达分析 1,DNA分子标记;2,紧密连接蛋白-3基因;3,紧密连接蛋白-4基因;4,β-肌动蛋白基因。

Fig.6 Gene expression analysis of pigeon intestine epithelium cells 1, DNA maker; 2, CLDN-3; 3, CLDN-4; 4, β-actin.

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鸽小肠上皮细胞的分离培养与鉴定

Isolation, culture and identification of pigeon intestine epithelium cells

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